Recent successes of immunotherapy have demonstrated the power of T cells to reject tumors. Two key observations have emerged from clinical studies. First, some patients with metastatic disease have a sufficient number and repertoire of tumor-reactive T cells that can be unleashed by immune checkpoint inhibitors to cause tumor regression. Second, unique mutated proteins expressed by an individual tumor are an untapped source of powerful tumor-specific T cell antigens. Because most cancer patients lack significant anti-tumor immune responses and do not respond to currently available immunotherapies, there is an urgent need to devise novel strategies to vaccinate these patients against their own individual tumor. We have pioneered studies to explore the use of local tumor radiotherapy (RT) as a means to release tumor antigens in an immunogenic context. We were the first to demonstrate that RT converted an insensitive mouse carcinoma into one responsive to CTLA-4 blockade, and have recently completed a prospective clinical trial testing this combination in lung cancer, a tumor type unresponsive to anti-CTLA-4 monotherapy. Interim analysis shows encouraging activity of the combination, with some complete and partial responses. Importantly, we have shown that priming of CD8 T cells specific for endogenous tumor antigens by RT requires immune checkpoint blockade. Our preliminary data also indicate that unique changes in T cell receptor (TCR) repertoire of intra-tumoral CD8 T cells are induced by RT + CTLA-4 blockade. Significant changes in TCR repertoire were also seen in peripheral blood of responding patients. Thus, our published and preliminary data strongly support the hypothesis that RT can generate an effective individualized in situ tumor vaccine. However, several unanswered questions hinder rapid progress in the use of RT as a widely available and relatively inexpensive strategy to target the patient-specific neoantigen repertoir. For instance, evidence that RT induces T cell responses to neoantigens is lacking, and it is not known if by changing the transcriptome of surviving irradiated cancer cells RT may expose unique neoantigens not expressed in untreated tumors. The influence of the RT regimen and of the immune checkpoint inhibitor used on this process remain poorly understood. Finally, while there is evidence that abscopal effects are mediated by T cells, the specificity of these T cell responses has not been characterized. Studies proposed are aimed at answering these questions in order to move the field forward and improve clinical trial design and the use of RT in combination with immunotherapy. A comprehensive evaluation of the specificity of T cells activated by RT combined with anti-CTLA-4 or anti-PD-1 will be performed. In addition, we will characterize the effects of RT on the tumor mutanome to identify potential immunogenic mutations exposed by RT. Analysis of samples from patients treated with RT+anti-CTLA-4 will provide preliminary evidence in humans.